Recirculating Memory T Cells Are a Unique Subset of CD4+ T Cells with a Distinct Phenotype and Migratory Pattern

Shannon K. Bromley, Sha Yan, Michio Tomura, Osami Kanagawa and Andrew D. Luster
J Immunol February 1, 2013, 190 (3) 970-976; DOI: https://doi.org/10.4049/jimmunol.1202805

Abstract

Several populations of memory T cells have been described that differ in their migration and function. In this study, we have identified a unique subset of memory T cells, which we have named recirculating memory T cells (TRCM). By exposing Kaede transgenic mouse skin to violet light, we tracked the fate of cutaneous T cells. One population of memory CD4+ T cells remained in the skin. A second population migrated from the skin into draining lymph nodes (LNs) in a CCR7-dependent manner. These migrating CD4+ T cells expressed a novel cell surface phenotype (CCR7int/+CD62LintCD69−CD103+/− E-selectin ligands+) that is distinct from memory T cell subsets described to date. Unlike memory T cell subsets that remain resident within tissues long-term, or that migrate either exclusively between lymphoid tissues or into peripheral nonlymphoid sites, CD4+ TRCM migrate from the skin into draining LNs. From the draining LNs, CD4+ TRCM reenter into the circulation, distal LNs, and sites of non-specific cutaneous inflammation. In addition, CD4+ TRCM upregulated CD40L and secreted IL-2 following polyclonal stimulation. Our results identify a novel subset of recirculating memory CD4+ T cells equipped to deliver help to both distal lymphoid and cutaneous tissues.

Introduction

Naive T cells migrate through lymphoid tissues, where they scan dendritic cells for cognate Ag. Upon Ag recognition, naive T cells are activated, and they expand and differentiate into effector T cells that can migrate into inflamed peripheral tissues. The majority of effector T cells then die by apoptosis. However, a heterogeneous pool of memory T cells survives to provide local and systemic protection in case of pathogen reexposure.

Early studies examining memory T cells in human blood identified two distinct memory T cell subsets that could be distinguished by their homing and effector capacities (1). By definition, central memory T cells (TCM) express lymph node (LN) tissue homing receptors and circulate exclusively between the blood and secondary lymphoid tissues. In contrast, effector memory T cells (TEM) migrate from the blood into nonlymphoid peripheral tissues. However, with the recent discovery of resident memory T cells (TRM) (2–4), we now know that the division of memory T cell subsets is more complex than initially appreciated. Memory T cells within extralymphoid tissues can include both TEM and TRM. Both of these memory T cell subsets lack CCR7 expression and are unable to reenter normal draining LNs. Although TRM can provide rapid response to pathogen rechallenge at a local site, depending on the stimulus, these cells migrate little and may be ineffective at providing protection at a distal site of Ag exposure (3, 5).

Although it has been suggested that the majority of cutaneous T cells is resident within the skin (2), several data suggest that a subset of memory T cells exits from the skin and reenters draining LNs (6–8). For example, large numbers of T cells have been identified in the afferent lymph of sheep. Although the majority of these T cells were found to express CD4, their phenotype and migratory fate remain incompletely defined (6). These recirculating memory T cells may provide extensive immune surveillance, delivering help not only at an initial cutaneous site of Ag challenge, but also within distal tissues. In support of this possibility, a recent study determined that memory CD4+ T cells migrated rapidly within the dermis and out of skin explant cultures (9), whereas memory CD8+ T cells were immobile within the skin following infection with HSV. Accordingly, CD4+ T cells but not CD8+ T cells were required to provide protection against reinfection at a site distal to the primary inoculation site (9). Although this study did not conclusively demonstrate that the CD4+ T cells that mediate this protection originated from the primary skin site, it did suggest that nonresident memory CD4+ T cells contribute to cutaneous immune protection. Therefore, elucidating the receptors expressed by recirculating memory CD4+ T cells, and defining their migration potential, may be beneficial for the study of memory CD4+ T cell responses.

Previous studies have demonstrated that adoptively transferred splenocytes or in vitro–activated T cells exit from peripheral tissues and enter into draining LNs in a CCR7-dependent manner (10, 11). However, these adoptively transferred cells might not reflect the cell populations present in the skin. In this study, we determined that endogenous memory CD4+ T cells exit from the skin in a CCR7-dependent manner. We identified their surface receptor phenotype, migratory fate, and function, and we suggest that they are a distinct memory T cell subset.

Materials and Methods

Mice

Kaede transgenic mice were obtained from Dr. Osami Kanagawa (RIKEN Institute) (12), rederived at Taconic, and then bred at Massachusetts General Hospital. CCR7-deficient (13) and C57BL/6 mice were obtained from the Jackson Laboratory. Mice were housed in a specific pathogen-free microisolator environment. Animal studies were approved by the institutional care and use committee at Massachusetts General Hospital.

Photoconversion of mice

Kaede transgenic mice were anesthetized, and a patch of abdominal skin (∼2 × 2 cm) was shaved. All remaining hair within the shaved patch was removed by applying Veet or Nair for 1 min, followed by washing with PBS. The shaved abdominal skin was exposed to violet light (420 nm) using a Bluewave LED visible light curing unit (Dymax) equipped with a 420-nm bandpass filter (Andover) with the light source at maximum power, 7.5 cm away from the skin. As reported previously, exposure of the shaved skin to violet light did not induce IL-1β mRNA expression, affect lymphocyte proliferation in response to polyclonal stimulation, or diminish CD4+ T cell chemotaxis to the chemokine CCL21 (8).

Tissues were isolated 24 h after photoconversion of abdominal skin for analysis, except where noted. For experiments analyzing the reentry of cutaneous T cells into the circulation and distal tissues, mice were photoconverted on 3 consecutive days and tissues were isolated 48 h after the final photoconversion. Similarly, mice were photoconverted on 3 consecutive days, and draining LNs were isolated after 8 d for analysis of the persistence of CD44, CCR7, CD62L, CCR4, and E-selectin ligands expression.

Bone marrow chimeras

Recipient CD45.1 mice were irradiated with 1000 rad prior to bone marrow injection. Single-cell suspensions of hind leg bone marrow from wild type (WT) CD45.1 CD45.2 mice, WT CD45.2 mice and CCR7-deficient CD45.2 mice were prepared. Recipient mice were injected with either a 1:1 mix of 5 × 106 WT CD45.1 CD45.2 and 5 × 106 CCR7-deficient CD45.2 bone marrow cells, or with a 1:1 mix of 5 × 106 WT CD45.1 CD45.2 and 5 × 106 WT CD45.2 bone marrow cells via lateral tail vein injection. After 8 wk, analysis of cells in blood and lymph nodes demonstrated that mice were reconstituted with both WT and CCR7-deficient T cells, with fewer CCR7-deficient than WT CD4+ T cells in LNs, but more in blood.

Abs and flow cytometry

Recovered tissue leukocytes were incubated for 20 min with TruStain FcX (BioLegend) and then stained with fluorochrome-conjugated Abs. The following mAbs were used: anti-mouse CD3, CD4, CD44, CD62L, CD103, CD69, CCR4, CCR7, and CCR9 (BioLegend or eBioscience). Staining for E-selectin ligands was performed using a chimeric E-selectin-Fc fusion protein (R&D Systems) as described (14), with allophycocyanin-conjugated anti-human IgG Fc-specific (Jackson ImmunoResearch) as the secondary staining reagent. All samples were stained with 7-AAD before acquiring data on an LSRII (BD Biosciences) to exclude dead cells from analysis. Data were analyzed using FlowJo (Tree Star). Mean fluorescence intensity (MFI) was determined by subtracting the MFI of the fluorescence minus one control from the MFI of the stained sample.

Isolation of cells from tissues

Blood (0.5 ml) was collected from mice by cardiac puncture. Mononuclear cells were then isolated by centrifugation over lymphocyte-mammal density gradients (Cedarlane Labs). Abdominal skin was excised, scraped to remove fat, minced, and digested in 5 ml HBSS 1% FBS containing 154 U/ml collagenase type IV (Sigma) at 37°C for 45 min. Samples were then vortexed for 30 s and washed through a 70-μm nylon filter. Draining axillary LNs and nondraining cervical LNs were isolated and processed, and recovered live cells were counted as described (11).

Cytokine secretion assay

Twenty-four hours after Kaede transgenic mouse abdominal skin photoconversion, draining axillary LNs were isolated and recovered cells cultured at 107 cells/ml in RPMI 1640 5% FBS in 24-well plates coated with anti-mouse CD3 (2 μg/ml; BioLegend) and addition of anti-mouse CD28 (1 μg/ml; BioLegend) for 4 h at 37°C. TAPI-2 (100 μM; Peptides International) was added to each well to preserve CD62L expression during stimulation. Cells were then analyzed for secretion of IL-2, IL-10, and IFN-γ using the appropriate cytokine secretion assay-detection kits (allophycocyanin; Miltenyi Biotec) following the manufacturer’s instructions.

CD40L staining

Twenty-four hours after Kaede transgenic mouse abdominal skin photoconversion, draining axillary LNs were isolated, and CD4+ T cells were purified using CD4 Dynabeads (Dynal) according to the manufacturer’s instructions. Purified CD4+ T cells were then cultured at 106 cells/ml in RPMI 1640 5% FBS in 24-well plates with or without PMA (50 ng/ml) and ionomycin (500 ng/ml) stimulation for 2 h at 37°C. Cells were then removed from plates, washed extensively, and costained with allophycocyanin-conjugated Ab directed against mouse CD40L (BioLegend).

Statistics

Comparisons were analyzed for statistical significance by Student t test with Microsoft Excel software, with p < 0.05 being considered significant.

Results

CCR7-dependent lymphocyte egress from normal skin

Using Kaede transgenic mice, we have measured the exit of endogenous lymphocyte subsets from the skin in vivo. The abdominal skin of Kaede mice was exposed to violet light for 5 min to induce the irreversible photoconversion of cutaneous cells (Fig. 1A). Initially, lymphocytes within the skin emit green fluorescence. However, immediately after exposure to violet light, cutaneous cells present within the skin during the 5-min period of photoconversion stably emit red fluorescence (8) (Fig. 1B). In this manner, we were able to track red fluorescent cells originating in the skin and migrating to the draining LN. Twenty-four hours after abdominal skin photoconversion, the draining axillary LNs, blood, and nondraining cervical LNs were isolated and recovered. Lymphocytes were counted, stained with Abs directed against CD3, CD4, CD8, and CD19, and analyzed by flow cytometry to identify exited Kaede-red+ cells among CD4+, CD8+, and CD19+ lymphocytes. Significantly more CD4+ T cells than CD8+ T cells or CD19+ B cells circulate from the skin to draining LNs (Fig. 1C, 1D). These data demonstrate that endogenous lymphocytes exit from the skin at baseline and correlate with recent studies demonstrating that more CD4+ T cells than CD8+ T cells migrate out of skin in explant cultures (9) and in T cell adoptive transfer studies (10).

FIGURE 1.
FIGURE 1.

Lymphocyte migration from skin to draining LNs in the steady state is CCR7 dependent. (A) Schematic of Kaede transgenic mouse photoconversion. The shaved abdominal skin of Kaede transgenic mice is exposed to 420-nm light for 5 min. After 24 h, leukocytes are recovered from tissues and analyzed by flow cytometry for CD3, CD4, CD8, CD19, and Kaede-green or Kaede-red expression. (B) Flow cytometric analysis of Kaede-green and Kaede-red expression by CD3+CD4+ T cells recovered from unconverted skin or photoconverted skin, blood, and draining axillary LN immediately after exposure to violet light. (C) Flow cytometric analysis of Kaede-green and Kaede-red expression by draining axillary LN CD3+CD4+ T cells, CD3+CD8+ T cells and CD3−CD19+ B cells 24 h after photoconversion of Kaede transgenic mouse abdominal skin. Data are representative of three experiments with six mice total. (D) Numbers of Kaede-red CD3+CD4+ T cells, CD3+CD8+ T cells, and CD3−CD19+ B cells within draining axillary LNs (left) or blood (right) 24 h after photoconversion of WT (black bars) or CCR7-deficient (gray bars) Kaede transgenic abdominal skin. Open bars are lymphocytes that fall within the Kaede-red gate from draining axillary LNs or blood of control unconverted Kaede transgenic mice. Data are from three experiments with six mice total for each genotype. A total of three control unconverted Kaede transgenic mice were analyzed. (E) The shaved abdominal skin of WT or CCR7-deficient mice was isolated, frozen in OCT, and 10-μm sections were stained with anti-CD4 Abs. Slides were imaged (original magnification ×200), and numbers of CD4+ T cells were counted. Data are the average of four to six mice, with at least three fields counted per mouse. (F) Five million WT CD4+ Thy1.1+ T cells were injected s.c. into the right footpads of either WT congenic Thy1.2+ or CCR7-deficient Thy1.2+ hosts. After 16 h, right draining popliteal LNs (DLN) and left contralateral popliteal LNs (non-DLN) were isolated. Recovered cells were counted, stained with anti-CD3, anti-CD4, and anti-Thy1.1 Abs and analyzed with flow cytometry. Data are from three experiments with nine mice total per genotype. (G) Bone marrow chimeras were generated by reconstituting irradiated WT CD45.1 mice with a 1:1 mix of either WT CD45.1 CD45.2 and CCR7-deficient CD45.2 bone marrow cells or with WT CD45.1 CD45.2 and WT CD45.2 bone marrow cells. After 8 wk, leukocytes were recovered from skin and the percentage of WT or CCR7-deficient CD3+ CD4+ T cells of total cutaneous CD3+ CD4+ T cells was calculated. Data are compiled from three experiments with WT:CCR7-deficient chimeras (left graph) and WT:WT chimeras (right graph) and include 11 or 8 chimeric mice total, respectively.

To determine whether the exit of resting endogenous lymphocytes from the skin is CCR7 dependent, we photoconverted the abdominal skin of WT Kaede and CCR7-deficient Kaede mice. After 24 h, we quantitated numbers of Kaede-red WT and Kaede-red CCR7-deficient CD4+ T cells, CD8+ T cells, and CD19+ B cells recovered from draining axillary LNs. Significantly more Kaede-red WT than Kaede-red CCR7-deficient CD4+, CD8+, and CD19+ cells were detected in draining LNs (Fig. 1D). The difference in accumulation of Kaede-red WT and Kaede-red CCR7-deficient lymphocytes within the draining LN does not appear to be due to increased exit of Kaede-red CCR7-deficient lymphocytes from the LN (15), because we did not detect increased numbers of Kaede-red CCR7-deficient lymphocytes compared with Kaede-red WT lymphocytes within the blood (Fig. 1D). In addition, we detected an equal number of CD4+ T cells in skin sections of CCR7-deficient mice compared with WT mice (Fig. 1E), suggesting that CCR7-deficient CD4+ T cells were present within the skin but failed to exit. Furthermore, the lack of CCR7-deficient lymphocyte egress from the skin is not explained by abnormal LN architecture in CCR7-deficient mice (13), because adoptively transferred WT CD4+ T cells migrated equally from the skin to draining LNs of WT and CCR7-deficient mice (Fig. 1F). To further demonstrate that the decreased exit of CCR7-deficient T cells from the skin is a result of the lack of CCR7 on T cells and not the result of other differences between WT and CCR7-deficient host mice, we generated WT/CCR7-deficient mixed bone marrow chimeras. In this competitive setting, a greater percentage of endogenous CCR7-deficient CD3+ CD4+ T cells than WT CD3+ CD4+ T cells accumulated within the skin of the same WT host (Fig. 1G). Collectively, these data suggest that the exit of endogenous CD4+, CD8+, and CD19+ lymphocytes from the skin requires CCR7.

CCR7−CD103+CD69+CD4+ T cells remain in skin

TRM cells have been identified in skin (2, 3, 9, 16) and are characterized by high CD103 and CD69 surface expression and a lack of CCR7 expression. In this study, we examined whether CD4+ T cells that remain in the skin following photoconversion also express these cell surface receptors. Because we determined that CCR7 regulates the exit of endogenous CD4+ T cells from the skin (Fig. 1D), we first examined whether we could identify differences in CCR7 surface expression on CD4+ T cells that remained in the skin 24 h after photoconversion versus CD4+ T cells that migrated out of the skin and into draining LNs. We observed that ∼40% of CD4+ T cells in normal mouse skin express CCR7. This percentage of CCR7+ T cells is similar to previous investigations demonstrating that ∼50% of T cells within normal human skin express CCR7 (2). Within 24 h after photoconversion, Kaede-green cells (which likely entered the area of photoconverted skin between the time of photoconversion and tissue harvest either from the blood or from adjacent, unconverted skin) included both CCR7+ and CCR7−CD4+ T cells. In contrast, few Kaede-red CCR7+ T cells are detected within the skin, suggesting that CCR7+ T cells exit the tissue efficiently. Whereas Kaede-red CD4+ T cells remaining in the skin were CCR7− (Fig. 2A), Kaede-red CD4+ T cells that migrated from the skin to draining LN expressed intermediate CCR7 (Fig. 2B). These results suggest that CCR7−CD4+ T cells are retained in the skin, whereas CCR7+CD4+ T cells are able to exit into the afferent lymphatic vessels and draining LNs.

FIGURE 2.
FIGURE 2.

Kaede-red CD4+ T cells residing in skin are CCR7−CD103+CD69+. Flow cytometric analysis of CCR7, CD103, and CD69 expression by Kaede-green CD3+CD4+ T cells (green solid line) and Kaede-red CD3+CD4+ T cells (red solid line) remaining within the abdominal skin (A) or migrated from skin to draining LNs (B) 24 h after abdominal skin photoconversion. Dashed lines are control “fluorescence minus one” stains of Kaede-green- and Kaede-red CD3+CD4+ T cells. Graphs depict the average percent of Kaede-green (green bars) and Kaede-red (red bars) CD3+CD4+ T cells in the skin or draining LNs that express CCR7, CD103, or CD69. For CCR7 expression in the draining LN, striped bars depict the percent of CD3+CD4+ T cells that express intermediate CCR7 and solid bars show the percent of CD3+CD4+ T cells that express high CCR7. Data are representative of three experiments with nine mice total.

In parallel, we determined whether Kaede-red CD4+ T cells remaining in the skin following photoconversion expressed higher levels of CD103 or CD69 than the Kaede-red CD4+ T cells that exited from the skin. CD103 binds to E-cadherin, which is expressed mainly by epithelial cells (17). Several studies suggest that CD103 retains T cells in tissue epithelium (18–20). In this study, we determined that Kaede-red CD4+ T cells remaining in the skin after photoconversion express CD103, which is consistent with a role for this molecule in the persistence of CD4+ T cells within the skin (Fig. 2A). However, we observed bimodal CD103 expression on CD4+ T cells that migrated from the skin to draining LNs (Fig. 2B), suggesting that CD103 expression alone is not a reliable marker for cutaneous resident memory CD4+ T cells, because it is also expressed on CD4+ T cells that exit from the skin.

The chemoattractant receptor S1P1 regulates the exit of T cells from thymic and LN tissues (21, 22). In addition, S1P1 can regulate T cell exit from peripheral tissues. Treatment of mice with FTY720 downregulates S1P1 expression and function, resulting in a partial reduction in CD4+ T cell exit from the skin (23). CD69 suppresses S1P1 function, thereby promoting the retention of activated (24) and naive (25) T cells within LNs. CD69 expression has also been detected on CD8+ TRM in the skin (5) and could act similarly to retain T cells within peripheral tissues. In this study, we examined CD69 expression on Kaede-red CD4+ T cells remaining in the skin 24 h after photoconversion. Whereas Kaede-red CD4+ T cells remaining in skin express CD69 (Fig. 2A), CD4+ T cells that migrate to draining LNs lack CD69 expression (Fig. 2B). These findings are consistent with a role for CD69 in the persistence of CD4+ T cells within the skin. These results suggest that a CCR7−CD103+CD69+ phenotype identifies CD4+ T cells that fail to exit from the skin.

Recirculating memory T cells express both LN- and skin-homing receptors

Distinct populations of memory T cells have been defined based on their homing receptor expression and function (1). TCM express the LN homing receptors CCR7 and CD62L, and thus recirculate through secondary lymphoid tissues. In contrast, TEM lack CCR7 and CD62L but express receptors for migration into peripheral tissues. To define the homing receptors expressed by T cells that exit the skin, we isolated lymphocytes from the draining axillary LNs 24 h after photoconversion of the abdominal skin. Recovered cells were stained with Abs directed against CD3, CD4, the memory marker CD44, and tissue-specific homing receptors and analyzed by flow cytometry. Almost all Kaede-red CD4+ T cells that migrated from the skin to draining LNs were CD44hiCCR7intCD62Lint. In addition, these cells express the skin homing receptors CCR4 and E-selectin ligands, but lack expression of the intestine homing receptor CCR9 (Fig. 3A, 3B).

FIGURE 3.
FIGURE 3.

Kaede-red CD4+ T cells express lymph node- and skin-homing receptors. (A) The top panel illustrates Kaede-green and Kaede-red expression by CD3+CD4+ T cells within the draining LN 24 h after photoconversion of Kaede transgenic mouse abdominal skin. The bottom panels depict flow cytometric analysis of CD44, CCR7, CD62L, E-selectin ligands, CCR4, and CCR9 expression by Kaede-green CD3+CD4+ (green solid line) and Kaede-red CD3+CD4+ T cells (red solid line) within the draining LNs 24 h after photoconversion of Kaede transgenic mouse abdominal skin. Dashed lines are control “fluorescence minus one” stains of Kaede-green- and Kaede-red CD3+CD4+ T cells. (B) MFI for receptor expression on Kaede-green CD3+CD4+ T cells and Kaede-red CD3+CD4+ T cells are displayed for one experiment with three mice per receptor analyzed and is representative of three experiments with at least eight mice total per receptor.

We next examined whether this CCR7intCD62Lint phenotype persists, or whether recirculating memory T cells (TRCM) convert to a TCM or TEM phenotype with time. Eight days after photoconversion, the recirculating memory CD4+ T cells upregulate CCR7, but remain CD62Lint (Fig. 4A, 4B). In addition, these CD4+ memory T cells maintain expression of the skin homing receptors E-selectin ligands and CCR4 (Fig. 4A, 4B). These results suggest that unlike TCM and TEM, recirculating CD4+ memory T cells have the potential to migrate through cutaneous and lymphoid tissues.

FIGURE 4.
FIGURE 4.

Kaede-red CD4+ T cells upregulate CCR7 expression over time. Shaved abdominal skin of Kaede transgenic mice was exposed to 420-nm light for 5 min on 3 consecutive days. (A) The top panel illustrates Kaede-green and Kaede-red expression by CD3+CD4+ T cells within the draining LNs 8 d after the final photoconversion of Kaede transgenic mouse abdominal skin. The bottom panel depicts flow cytometric analysis of CD44, CCR7, CD62L, E-selectin ligands, and CCR4 expression on these Kaede-green CD3+CD4+ (green solid line) and Kaede-red CD3+CD4+ T cells (red solid line). Dashed lines are control “fluorescence minus one” stains of Kaede-green- and Kaede-red CD3+ CD4+ T cells. (B) MFI for receptor expression on Kaede-green CD3+CD4+ T cells and Kaede-red CD3+CD4+ T cells are displayed for one experiment with three mice per receptor analyzed and is representative of three experiments with at least eight mice per receptor.

TRCM reenter the circulation and distal tissue

The homing receptor expression on Kaede-red CD4+ T cells that migrate from skin to draining LNs suggests that they circulate between these tissues. Therefore, we examined whether we could identify CD4+CD44hi E-sel-ligands+ CD62LintCCR7int/+ T cells within the circulation at steady state. We detected E-sel-ligands+ memory T cells with intermediate/+ expression of CCR7 and CD62L in the blood of resting C57BL/6 mice (Fig. 5A). In parallel, we examined whether the Kaede-red CD4+ memory T cells that have migrated from skin to draining LNs subsequently enter the circulation. To address this question, we photoconverted the abdominal skin of Kaede transgenic mice every 24 h for 3 d. We photoconverted the skin multiple times to generate sufficient numbers of Kaede-red CD4+ T cells to track. Forty-eight hours after the final photoconversion, we isolated the blood, draining axillary LNs, and nondraining cervical LNs. Next, recovered leukocytes were analyzed by flow cytometry to detect Kaede-red CD4+ T cells. Kaede-red CD4+ T cells were detected in the blood, demonstrating that these memory T cells exit from draining LNs and reenter the circulation (Fig. 5B). In addition, these CD4+ TRCM were detected in nondraining cervical LNs (Fig. 5B). Therefore, despite our finding that these cells express only intermediate CD62L, they are still able to gain entry into distal LNs, either via HEV or afferent lymphatic vessels draining distal skin sites. However, we were unable to identify Kaede-red CD4+ T cells within normal distal ear skin. The skin is a large tissue through which the migrating T cells can disperse. Nonetheless, because these memory CD4+ T cells enter the circulation and express the skin-homing receptors E-selectin ligands and CCR4, it remained possible that they were able to migrate into normal skin, but were present in numbers too low to recover and detect. Indeed, studies in sheep demonstrate that T cells isolated from normal skin-draining afferent lymph and injected directly into the bloodstream migrate back into skin (26). Consistent with this finding, we detected recirculating memory CD4+ T cells within a distal site of nonspecific cutaneous inflammation induced by injecting the ear with CFA (Fig. 5C). These results suggest that unlike TCM, TEM or TRM, these TRCM are able to migrate between distal lymphoid and cutaneous sites to provide widespread cutaneous immune surveillance.

FIGURE 5.
FIGURE 5.

Kaede-red CD4+ T cells reenter the circulation from draining LNs. (A) CCR7 and CD62L expression on CD4+CD44hi E-sel-Fc+ T cells within the blood of resting C57BL/6 mice. Leukocytes recovered from the blood were gated on CD3+CD4+CD44hi memory T cells and on CD3+CD4+CD44lo-int naive T cells (top left panel). The CD44hi T cell population was then gated on E-sel-Fc+ cells (top right panel). The middle and bottom histograms depict CCR7 and CD62L expression on CD4+CD44lo-int naive T cells, CD4+CD44hi bulk memory T cells, and CD4+CD44hi E-sel-Fc+ memory T cells. Each plot is from six mice pooled and is representative of three independent experiments. Graphs depict the average percent of each CD3+CD4+ T cell population in the blood that express CCR7 or CD62L. Black bars depict the percent of CD3+CD4+ T cells that express intermediate CCR7 or CD62L, and white bars show the percentage of CD3+CD4+ T cells that express high CCR7 or CD62L. (B) Shaved abdominal skin of Kaede transgenic mice was exposed to 420-nm light for 5 min on 3 consecutive days. Flow cytometric analysis of Kaede-green and Kaede-red expression on CD3+CD4+ T cells recovered from the blood, nondraining cervical LNs, and draining axillary LNs 48 h after the final photoconversion. Data are representative of three experiments with eight mice per group. (C) The right ear of Kaede transgenic mice was injected with 10 μl of a 1:1 PBS:CFA emulsion, and the shaved abdominal skin was photoconverted as in (A). Flow cytometry plots depict Kaede-green and Kaede-red expression by CD3+CD4+ T cells recovered from the CFA-injected distal ear skin 48 h after the final photoconversion. Numbers in plots are average percent Kaede-red T cells of CD3+CD4+ T cells recovered from the ear and are calculated from three experiments with 8–14 mice per group. Data are representative of six mice pooled per group.

TRCM secrete IL-2 and upregulate CD40L

Previous studies demonstrated that CD4+ TCM and TEM cells display distinct effector functions. Whereas TCM produce IL-2 and only negligible IFN-γ, TEM cells secrete IFN-γ after stimulation (1). To determine the effector functions mediated by recirculating memory CD4+ T cells, we examined their cytokine secretion following in vitro stimulation with plate-bound anti-CD3 and soluble anti-CD28. Both central memory Kaede-green CD4+ T cells and recirculating memory Kaede-red CD4+ T cells secreted IL-2, but negligible IFN-γ and IL-10 (Fig. 6A). In addition, both Kaede-green and Kaede-red memory CD4+ T cells upregulated CD40L expression upon activation (Fig. 6B). Based on their cytokine secretion and CD40L expression, CD4+ TRCM resemble TCM. However, unlike TCM that circulate only between lymphoid tissues, TRCM express chemokine receptors and adhesion molecules that enable their delivery of help to both lymphoid and nonlymphoid tissues.

FIGURE 6.
FIGURE 6.

Recirculating Kaede-red CD3+CD4+CD44hi T cells upregulate CD40L and secrete IL-2. Shaved abdominal skin of Kaede transgenic mice was exposed to 420-nm light for 5 min. After 24 h, draining LNs were isolated and (A) recovered cells were stimulated for 4 h with plate-bound anti-CD3 and soluble anti-CD28. Cytokine secretion by Kaede-green CD3+CD4+CD44hiCD62Lhi memory T cells and Kaede-red CD3+CD4+CD44hi memory T cells was analyzed using flow cytometry. Data are pooled from three experiments with nine mice total for each cytokine. (B) CD4+ T cells were purified from draining LNs and stimulated for 2 h with PMA and ionomycin or left untreated. Next, surface CD40L expression was analyzed on Kaede-green CD44lo naive T cells (gray lines), Kaede-green CD44hi memory T cells (green lines), or Kaede-red CD44hi memory T cells (red lines). Data are representative of two experiments with six mice total.

Discussion

Using Kaede transgenic mice, we have tracked the migration of endogenous cutaneous lymphocytes in vivo. We have found that one population of CD4+ T cells remains in the skin after photoconversion and is CD44hiCD69+CD103+. In addition, we have identified a unique population of memory CD4+ T cells whose phenotype and migratory pattern differs from TCM, TEM, and TRM. These CD4+ TRCM migrate from normal skin to draining LNs in a CCR7-dependent manner. From the draining LNs, they reenter the circulation, distal LNs, and sites of nonspecific cutaneous inflammation. Following polyclonal stimulation, these memory T cells upregulate CD40L and secrete IL-2. Our results identify a recirculating memory CD4+ T cell subset equipped to deliver help to both distal lymphoid and cutaneous tissues.

Although previous studies have identified a population of resident memory T cells within the skin (2, 3), our results suggest the presence of a subset of memory T cells that is present only transiently within the skin, displaying dynamic migration through the skin and into afferent lymph. We have observed that ∼40% of CD4+ T cells in normal mouse skin express CCR7. Within 24 h after photoconversion, few Kaede-red CCR7+ T cells are detected within the skin. However, CCR7int cells originating from the skin are detected in draining LN, suggesting that CCR7+ T cells exit the tissue efficiently. A prior investigation revealed that adult human skin contains ∼2 billion memory T cells (2). More than 50% of these cutaneous memory T cells express CCR7 (2), suggesting that a substantial number of human memory T cells also have the potential to exit from normal skin. Of note, CCR7+ memory T cells in human blood have been found to express tissue and inflammatory chemokine receptors, including CCR4 (1). It is possible that like the TRCM cells we have characterized in this study, these human CCR7+ memory T cells also migrate between cutaneous and lymphoid tissues.

We have determined that recirculating memory CD4+ T cells can be identified based on their surface receptor expression: CD44hi, CCR7int-pos, CD62Lint, CD103+/−, CD69−, CCR4+/−, and e-sel-ligands+. Previous studies have identified regulatory T cells with a similar cell surface phenotype that migrate from skin to draining LNs during a DNFB-mediated hypersensitivity response (8). Thus, this homing receptor expression profile may define both recirculating cutaneous memory CD4+ T cells and regulatory T cells at homeostasis and inflammation. Of note, although these recirculating memory CD4+ T cells are able to migrate into distal skin sites, these T cells lack expression of the intestine-homing receptor CCR9. Early studies performed in sheep have identified memory T cells in cutaneous lymph (7) and intestinal lymph (27), suggesting that separate populations of memory T cells exist, recirculate through additional tissues, and can be characterized by distinct homing receptor expression profiles.

Previous studies have discovered populations of memory T cells with distinct homing potentials (1). By definition, TCM cells express LN tissue homing receptors and circulate exclusively between the bloodstream and secondary lymphoid tissues. In contrast, TEM cells migrate from blood into extralymphoid tissues, but lack CCR7 expression, and so are unable to enter into resting LNs. Whether CD4+ TRCM are a subset completely distinct from TCM or TEM is unknown. Although CD4+ TRCM were CCR7int CD62Lint shortly after their exit from the skin, over time they upregulated CCR7 expression but remained CD62Lint, E-selectin ligands+, CCR4+/−. Based on this unique homing receptor expression profile and migratory pattern, we propose that they are a distinct memory T cell subset, but future experiments will determine the relationship of this subset to other memory T cell subsets.

In conclusion, our data identify a distinct population of memory CD4+ T cells that circulate between lymphoid and cutaneous tissues and that can be identified by their surface receptor expression. Reentry of memory T cells into the circulation might allow for surveillance and delivery of help distal to the site of initial Ag exposure (e.g., pathogen spread or reinfection). In this study, we have identified TRCM surface receptors that may be useful in the study of memory CD4+ T cell response and for the development of vaccination strategies requiring widespread surveillance of cutaneous tissues by T cells.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank members of the Luster laboratory for helpful discussions.

Footnotes

  • This work was supported by National Institutes of Health Grants R01-CA069212 (to A.D.L.) and K01-AR053715 (to S.K.B.) and by a Claflin Distinguished Scholar Award (to S.K.B.).

  • Abbreviations used in this article:

    LN
    lymph node
    MFI
    mean fluorescence intensity
    TCM
    central memory T cell
    TEM
    effector memory T cell
    TRCM
    recirculating memory T cell
    TRM
    resident memory T cell
    WT
    wild type.

  • Received October 5, 2012.
  • Accepted November 16, 2012.

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